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EJB Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.1 No.3, Issue of August 15, 1998. © 1998 by Universidad Católica de Valparaíso –- Chile Invited review paper / Received 5 September 1998

This paper is available on line at http://www.ejb.org

REVIEW ARTICLE

General and microbiological aspects of solid substrate fermentation

Maurice RaimbaultLaboratoire de Biotechnologie Microbienne Tropicale, Centre ORSTOM-LBMT 911 av. Agropolis - B.P.:5045 - 34032 Montpellier (France)

E-mail: [email protected]

http://www.mpl.orstom.fr

At first some general considerations about specificity

and characteristics of SSF, their advantages and disad-

vantages as compared to LSF, are presented.

Microorganisms involved in solid substrate

fermentations are identified, considering the better per-

formances of filamentous fungi. The solid substratesand their basic macromolecular compounds are detai-

led in relation to this complex and heterogeneous sys-

tem. Biomass measurement is examined in detail, as

well as environmental factors, both essential for

studying and optimising solid substrate fermentations.

General considerations

Aerobic microbial transformation of solid materials or "So-

lid Substrate Fermentation" (SSF) can be defined in terms

of the following properties:

- A solid porous matrix which can be biodegradable or not, but with a large surface area per unit volume, in the range

of 103 to 106 m2/ cm3, for a ready microbial growth on the

solid/gas interface.

- The matrix should absorb water amounting one or several

times its dry weight with a relatively high water activity on

the solid/gas interface in order to allow high rates of bio-

chemical processes.

- Air mixture of oxygen with other gases and aerosols

should flow under a relatively low pressure and mix the

fermenting mash.

- The solid/gas interface should be a good habitat for the

fast development of specific cultures of moulds, yeasts or bacteria, either in pure or mixed cultures.

- The mechanical properties of the solid matrix should

stand compression or gentle stirring, as required for a gi-

ven fermentation process. This requires small granular or

fibrous particles, which do not tend to break or stick to

each other.

- The solid matrix should not be contaminated by inhibi-

tors of microbial activities and should be able to absorb or

contain available microbial foodstuffs such as carbohy-

drates (cellulose, starch, sugars) nitrogen sources (ammo-

nia, urea, peptides) and mineral salts.

Typical examples of SSF are traditional fermentations such

as:

- Japanese "koji" which uses steamed rice as solid substrate

inoculated with solid strains of the mould Aspergillus ory-

zae.

- Indonesian "tempeh" or Indian "ragi" which use steamedand cracked legume seeds as solid substrate and a variety

of non toxic moulds as microbial seed.

- French "blue cheese" which uses perforated fresh cheese

as substrate and selected moulds, such as Penicillium ro-

quefortii as inoculum.

- Composting of lignocellulosic fibres, naturally contami-

nated by a large variety of organisms including cellulolytic

bacteria, moulds and Streptomyces sp.

- In addition to traditional fermentations, new versions of

SSF have been invented. For example, it is estimated that

nearly a third of industrial SSF and koji processes in Japan

has been modernised for large scale production of citric

and itaconic acids.

Furthermore, new applications of SSF have been suggested

for the production of antibiotics (Barrios et al., 1988),

secondaries metabolites (Trejo-Hernandez et al., 1992,

1993) or enriched foodstuffs (Senez et al., 1980).

Presently SSF has been applied to large-scale industrial

processes mainly in Japan. Traditional koji, manufactured

in small wooden and bamboo trays, has changed gradually

to more sophisticated processes: fixed bed room fermenta-

tions, rotating drum processes and automated stainless

steel chambers or trays with microprocessors, electronicssensors and servomechanical stirring, loading and dischar-

ging. The usual scale in sake or miso factories is around 1

or 2 metric tons per batch, but reactors can be made and

delivered by engineering firms to a capacity as large as 20

tons (Fujiwara, Ltd.).

Outside Japan, Kumar (1987) has reported medium scale

production of enzymes, such as pectinases, in India. Koji

type processes are widely used in small factories of the Far

East (Hesseltine, 1972) and koji fermentation has been

adapted to local conditions in United States (USA) and

other Western countries, including Cuba (III A). In France,

a new firm (Lyven S.A.) was recently created to


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Raimbault, M.

175

commercialise a process for pectinase production from su-

garbeet pulp. Blue cheese production in France is being

modernised with improvements on the mechanical condi-

tioning of cheeses, production of mould spores and control

of environmental conditions.

Composting, which was developed for small-scale produc-

tion of mushrooms, has been modernised and scaled up in

Europe and USA. Also, various firms in Europe and USA

produce mushroom spawn by cultivating Agaricus, Pleuro-

tus or Shii-Take aseptically on sterile grains in static

conditions.

New versions for SSF reactors have been developed in

France (Durand et al., 1988; Roussos et al., 1993, Durand

et al., 1997), Cuba (Cabello, 1985; Enríquez, 1983 and Ro-

dríguez, 1986), Chile (Fernández et al., 1996) and funda-

mental studies on process engineering are being conductedin Mexico (Saucedo-Castañeda, 1990).

SSF is a batch process using natural heterogeneous mate-

rials (Raimbault, 1981 and Tengerdy, 1985), containing

complex polymers like lignin (Agosin et al, 1989), pectin

(Kumar, 1987; Oriol, 1988a), lignocellulose (Roussos,

1985). SSF has been focused mainly to the production of

feed, hydrolytic enzymes, organic acids, gibberelins,

flavours and biopesticides.

Most of the recent research activity on SSF is being done

in developing nations as a possible alternative for conven-tional submerged fermentations, which are the main pro-

cess in pharmaceutical and food industries in industrialised

nations.

SSF seems to have theoretical advantages over liquid

substrate fermentation (LSF). Nevertheless, SSF has seve-

ral important limitations. Table 1 shows advantages and

disadvantages of SSF compared to LSF.

Table 2 presents a list of SSF processes in economical sec-

tors of agro-industry, agriculture and fermentation

industry. Most of the processes are commercialised in

South-East Asian, African, and Latin American countries. Nevertheless, a resurgence of interest has occurred in Wes-

tern and European countries over the last 10 years. The fu-

ture potentials and applications of SSF for specific proces-

ses are discussed later.

Table 1. Comparison between Liquid and Solid Substrate Fermentations.

FACTOR Liquid

Substrate Fermentation

Solid

Substrate Fermentation

Soluble Substrates (sugars) Polymer Insoluble Substrates:

Sub strates Starch Cellulose Pectines Lignin

Aseptic conditions Heat sterilisation and aseptic

Control

Vapor treatment, non sterile

conditions

Water High volumes ofwaterconsu-med

and effluents discarded

Limited Consumption of Wa-ter;

low Aw. No effluent

Metabolic Heating Easy control of temperature Low heat transfer capacity

Easy aeration and high surfa ce

exchange air/substrate

Aeration Limitation oby soluble oxygen High

level of air requiredpH control Easy pH control Buffered solid substrates

Mecanical agitation Good homogeneization Static conditions prefered

Scale up Industrial equipments Available Need for Engineering & New

design Equipment

Inoculation Easy inoculation , continuous

process

Spore inoculation, batch

Contamination Risks of contamination for single

strain bacteria

Risk of contamination for low rate

growth fungi

Energetic consideration High energy consuming Low energy consuming

Volume of Equipment High volumes and high cost

technology

Low volumes &low costs of

equipments


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Effluent & pollution High volumes of polluting effluents No effluents, less pollution

Concentration S /Products 30-80 g/1 100/300g/1

The following considerations summarise the present status

of SSF:

- Potentially many high value products, as enzymes, primary and secondary metabolites, could be produced in

SSF. But improvements in engineering and socio-econo-

mic aspects are required because processes must use cheap

substrates locally available, low technology applicable in

rural areas, and processes therefore must be simplified.

- Potential exists in developed countries, but close co-ope-

ration and exchange between developing and industrialised

countries are required for further application of SSF.

- The greatest socio-economical potential of SSF is the rai-

sing of living standards through the production of protein

rich foods for human consumption. Protein deficiency is a

major cause of malnutrition and the problem will become

worse with further increases in world population. Two

alternatives can be explored to tackle this problem:

- Production of protein-enriched fermented foods for directhuman consumption. This alternative involves starchy

substrates for its initial nutritional caloric value. Successful

production of such foods will require demonstration of eco-

nomical feasibility, safety, significant nutritional improve-

ment, and cultural acceptability.

- Production of fermented materials for animal feeding.

Starchy substrates protein-enriched by SSF could be fed to

monogastric animals or poultry. Fermented lignocellulosic

substrates, by increasing its fibre digestibility, could be fed

to ruminants. In this case, the economical feasibility

should be favourable in comparison to the common model

using protein of soybean cake, a by-product of soybean oil. product of soybean oil.

Table 2. Main applications of SSF processes in various economical sectors

Economical Sector Application Examples

Agro-Food Industry Traditional Food Fermentations Koji, Tcznpch, Rae, Attickc,

Fermented cheeses

Mushroom Production & spawn Agaricus, Pleurotus, Shn-take

Bioconversion By-products Sugar pulp Bagass Coffee pulp’

Silage Composting, Detoxication Food Additives Flavours. Dyestuffs. Essential Fat

and organic acids

Agriculture Biocontrol , Bioinsecticide Beauveria Metarhizium, Tricho

derma

PlantGrowth, Hormones Gioberellins, Rhizobium, In-choderma

Mycorhization, Wild Mushroom Plant inoctiation,

Industrial

Fermentation

Enzymes production Amylases, Cellulases Proteases,

Pectinases, Xylanases

Artibiotic prduction Penecillin, feed & Probiotics

Organic acid Production Ciric acid

Fumaric acidGallic acic

Lactic acid

Ethanol Prodixtion Schwanniomyces sp. Sbrch

Malting and Brewing

Fungal Metabolites Hormones Alcaloides,

For the last 15 years, the Orstom group has been working

on solid fermentation process for improving protein

content of cassava and other tropical starchy substrates

using fungi, specially from Aspergillus group, in order to

transform starch and mineral salts into fungal proteins

(Raimbault, 1981).


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177

More recently, C. Soccol working at our Orstom laboratory

in Montpellier, obtained good results with fungi of the

Rhizopus group, of special interest in human traditional

fermented foods (Soccol, 1992). Increasing knowledge

about specificity of strains of Rhizopus able to degrade

crude granules of starch has been recently gathered atOrstom, which will drastically simplify the process of SSF.

The ORSTOM group is also collaborating since 1981 with

a group at the Universidad Autónoma Metropolitana

(UAM) in Mexico on the following aspects:

- Protein enrichment of cassava and starchy substrates

- Production of organic acids or ethanol by SSF from star-

chy substrate and cassava

- Digestibility of fibres and lignocellulosic materials for

animal feeding

- Degradation of caffein in coffee pulp and ensiling for

conservation and detoxification

- Production of enzymes and fungal metabolites by SSFusing sugarcane bagasse

Hopefully in the future, an extended collaborative program

could be fitted for other Latin-American research groups

involved in SSF, which might originate an international

network including American, Asian, European and Austra-

lian groups.

Microorganisms

Bacteria, yeasts and fungi can grow on solid substrates,

and find application in SSF processes. Filamentous fungi

are the best adapted for SSF and dominate in researchworks. Some examples of SSF processes for each category

of micro-organisms are reported in Table 3.

Bacteria are mainly involved in composting, ensiling and

some food processes (Doelle et al., 1992). Yeasts can be

used for ethanol and food or feed production (Saucedo-

Castañeda et al., 1992a, 1992b).

But filamentous fungi are the most important group of

microorganisms used in SSF process owing to their physio-

logical, enzymological and biochemical properties.

The hyphal mode of fungal growth and their good toleran-

ce to low water activity (Aw) and high osmotic pressure

conditions make fungi efficient and competitive in natural

microflora for bioconversion of solid substrates.

Koji and Tempeh are the two most important applicationsof SSF with filamentous fungi. Aspergillus oryzae is grown

on wheat bran and soybean for Koji production, which is

the first step of soy sauce or citric acid fermentation. Koji

is a concentrated hydrolytic enzyme medium required in

further steps of the fermentation process. Tempeh is an In-

donesian fermented food produced by the growth of Rhizo-

pus oligosporus on soybeans. People consume the fermen-

ted product after cooking or toasting. The fungal fermenta-

tion allows better nutritive quality and degrades some anti-

nutritional compounds contained in the crude soybean.

The hyphal mode of growth gives a major advantage to fi-lamentous fungi over unicellular microorganisms in the

colonisation of solid substrates and for the utilisation of

available nutrients. The basic mode of fungal growth is a

combination of apical extension of hyphal tips and the ge-

neration of new hyphal tips through branching. An impor-

tant feature is that, although extension occurs only at the

tip at a linear and constant rate, the frequency of branching

makes the kinetic growth pattern of biomass exponential,

mainly in the first steps of the vegetative stage. That point

is important for growth modelling and will be discussed

further.

The hyphal mode of growth gives the filamentous fungi the power to penetrate into the solid substrates. The cell wall

structure attached to the tip and the branching of the myce-

lium ensure a firm and solid structure. The hydrolytic en-

zymes are excreted at the hyphal tip, without large dilution

like in the case of LSF, what makes the action of hydrolytic

enzymes very efficient and allows penetration into most so-

lid substrates. Penetration increases the accessibility of all

available nutrients within particles.

Table 3. Main groups of microorganisms involved in SSF processes.

Microflora SSF Process

Bacteria Bacillus sp. Composting, Natto, amylase

Pseudomonas sp. Composting Serratia sp. Composting

Streptoccus sp. Composting

Lactobacillus sp. Ensiling, Food Clostidrium sp. Ensiling, Food

Yeast Endomicopsis burtonii Tape, cassava, rice


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Saccharomyces cerevisiae Food, Ethanol

Schwanniomyces castelli Ethanol, Amylase

Fungi

Altemaria sp. Composting

Aspergillus sp. Composting, Industrial, Food Fusarium sp. Composting. GibberellinsMonilia sp. Composting

Mucor sp. Composting, Food; enzime

Rhizopus sp. Composting. Food, enzimes, organic acids

Phanerochaete chrysosporium Composting, lignin degradation

Trichoderma sp. Composting Biological control, BioinsecticideBeauveria sp., Metharizium sp. Biological control, Bioinsecticide Amylomyces rouxii Tape cassava, rice Aspergillus oryzae Koji, Food, citric acid Rhizopus oligosporus Tempeh, soybean, amylase, lipase

Aspergillus niger Feed, Proteins, Amylase, ctric acid Pleurotus oestreatus, sajor-caju Mushroom

Lentinus edodes Shii-take mushroomPenicilium notatum, roquefortii Penicillin, Cheese

Fungi cannot transport macromolecular substrates, but the

hyphal growth allows a close contact between hyphae and

substrate surface. The fungal mycelium synthetises and ex-

cretes high quantities of hydrolytic exoenzymes. The resul-

ting contact catalysis is very efficient and the simple pro-

ducts are in close contact to enter the mycelium across the

cell membrane to promote biosynthesis and fungal metabo-lic activities. This contact catalysis by enzymes can explain

the logistic model of fungal growth commonly observed

(Raimbault, 1981). That point will be discussed further.

Substrates

All solid substrates have a common feature: their basic ma-

cromolecular structure. In general, substrates for SSF are

composite and heterogeneous products from agriculture or

by-products of agro-industry. This basic macromolecular

structure (e.g. cellulose, starch, pectin, lignocellulose,

fibres etc..) confers the properties of a solid to the substra-te. The structural macromolecule may simply provide an

inert matrix (sugarcane bagasse, inert fibres, resins) within

which the carbon and energy source (sugars, lipids, orga-

nic acids) are adsorbed. But generally, the macromolecular

matrix represents the substrate and provides also the car-

bon and energy source.

Preparation and pre-treatment represent the necessary

steps to convert the raw substrate into a form suitable for

use, that include:

- size reduction by grinding, rasping or chopping

- physical, chemical or enzymatic hydrolysis of polymers to

increase substrate availability by the fungus.

- supplementation with nutrients (phosphorus, nitrogen,

salts) and setting the pH and moisture content, through a

mineral solution

- cooking or vapour treatment for macromolecular struc-ture pre-degradation and elimination of major contami-

nants. Pre-treatments will be discussed under individual

applications.

The most significant problem of SSF is the high heteroge-

neity, which makes difficult to focus one category of hydro-

lytic processes, and leads to poor trials of modelling. This

heterogeneity is of different nature:

- non-uniform substrate structure (mixture of starch, ligno-

cellulose, pectin)

- Variability between batches of substrates, limiting the

reproducibility

- Difficulty of mixing solid mass in fermentation, in order to avoid compaction, which causes non uniform growth,

gradients of temperature, pH and moisture, that makes

representative samples almost impossible to obtain.

Each macromolecular type of substrate presents different

kind of heterogeneity:

Lignocellulose occurs within plant cell walls, which

consist of cellulose microfibrils embedded in lignin, hemi-

cellulose and pectin. Each category of plant material

contains variable proportion of each chemical compound.

Two major problems can limit lignocellulose breakdown:


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Raimbault, M.

179

- cellulose exists in four recognised crystal structures

known as celluloses I, II, III and IV. Various chemical

or thermal treatments can change the structure from

crystalline to amorphous.

- - different enzymes are necessary in order degrade cel-

lulose, e.g. endo and exo-cellulases plus cellobiase

Pectins are polymers of galacturonic acid with different ra-

tio of methylation and branching. Exo-and endo pectinases

and demethylases that hydrolyse pectin into galacturonic

acid and methanol. Hemicelluloses are divided in major

three groups: xylans, mannans and galactans. Most of he-

micelluloses are heteropolymers containing two to four

different types of sugar residues.

Lignin represents between 26 to 29% of lignocellulose,

and is strongly bounded to cellulose and hemicellulose,

hiding them and protecting them from the hydrolase at-tack. Lignin peroxidase is the major enzyme involved in li-

gnin degradation. Phanerochaete chrysosporium is the

most recognised fungi for lignin degradation.

So, lignocellulose hydrolysis is a very complex process. Ef-

fective cellulose hydrolysis requires the synergetic action of

several cellulases, hemicellulases and lignin peroxidases.

Despite this, lignocellulose is a very abundant and cheap

natural renewable material, so a lot of work has been

conducted on its microbial breakdown, specially with fun-

gal species.

Starch is another very important and abundant natural so-

lid substrate. Many microorganisms are capable to hydro-

lyse starch, but generally its efficient hydrolysis requires

previous gelatinization. Some recent works concern the

hydrolysis of the raw (crude or native) starch as it occurs

naturally.

The chemical structure of starch is relatively simple com-

pared to lignocellulose substrates. Essentially starch is

composed of two related polymers in different proportions

according to its source: amylose (16-30%) and amylopectin

(65-85%). Amylose is a polymer of glucose linked by α -1,4 bonds, mainly in linear chains. Amylopectin is a large

highly branched polymer of glucose including also α-1,6 bonds at the branch points.

Within the plant, cell starch is stored in the form of granu-

les located in amyloplasts, intracellular organelles surroun-

ded by a lipoprotein membrane. Starch granules are highly

variable in size and shape depending on the plant material.Granules contain both amorphous and crystalline internal

regions in respective proportions of about 30/70. During

the process of gelatinization, starch granules swell when

heated in the presence of water, which involves the brea-

king of hydrogen bonds, especially in the crystalline re-

gions.

Many microorganisms can hydrolyse starch, specially fun-

gi which are then suitable for SSF application involving

starchy substrates. Glucoamylase, α-amylase, β-amylase, pullulanase and isoamylase are involved in the processes of

starch degradation. Mainly α-amylase and glucoamylaseare of importance for SSF.

α-amylase is an endo-amylase attacking α-1,4 bonds inrandom fashion which rapidly reduce molecular size of

starch and consequently its viscosity producing liquefac-

tion. Glucoamylase occurs almost exclusively in fungi in-

cluding Aspergillus and Rhizopus groups. This exo amyla-

se produces glucose units from amylose and amylopectin

chains.

Microorganisms generally prefer gelatinised starch. But

large quantity of energy is required for gelatinization so it

would be attractive to use organisms growing well on raw

(ungelatinised) starch. Different works are dedicated to

isolate fungi producing enzymes able to degrade raw

starch, as has been done by Soccol et al., (1994), Berg-

mann et al., (1988) and Abe et al., (1988).

In our laboratory, many studies concerning SSF of cassava,

a very common tropical starchy crop, have been conducted

with the purpose of upgrading protein content, both for

animal feeding using Aspergillus sp. and for direct human

consumption, using Rhizopus. Table 4 indicates the protein

enrichment with different fungi.fungi.

Table 4. Protein enrichment of Cassava by various selected strains of fungi. (Raimbault et al., 1985)

Inoculum Composition (% dry basis)

Strain SourceTime(h)

Protein Total Sugar

Aspergillus niger no.10 Cassava 25 16.5 35.6

Aspergillus awamori no.12 Koji 30 16.3 35.1

Aspergillus usamii no.M140 Koji 30 15.6 29.5 Monilia sitophila no. 27 Pozol 42 15.1 32.3


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Rhizopus sp. no. 7 Cassava 48 14.9 39.3

Aspergillus oryzae no.M84 Koji 30 14.8 30.0

Aspergillus sp. no. B1 Banana 30 14.7 39.1

Aspergillus sp. no. T1 Tempeh 30 14.3 34.0

Aspergillus niger no. 31 Cassava 30 14.3 34.5

Aspergillus sp. no. 14 Cassava 30 14.2 37.9 Aspergillus terricola no. R3 Ragi 30 14.1 10.9

Aspergillus sp. no. M101 Tempeh 30 14.0 31.4

Aspergillus sp. no. 72 Banana 30 13.8 28.2

Aspergillus awamori no. 13 Koji 48 13.0 38.8

Aspergillus sp. no. M147 Koji 30 12.7 32.4

Aspergillus niger no. 17 Cassava 30 12.0 45.2

Aspergillus sp. no. 39 Banana 30 11.1 40.0

Aspergillus sp. no. M82 Tempeh 30 10.9 38.0

Raw cassava -- -- 2.50 90.00

Recently good results were obtained by Soccol in the pro-

tein enrichment of cassava and cassava bagasse using se-

lected strains of Rhizopus. Biotransformed starchy flours

were produced containing 10-12% of good quality protein,

comparable to cereal. Such biotransformed cassava flour

can be used as cereal substitute for breadmaking up to a

level of 20%, without any perceivable change by the consu-

mer.

Biomass Measurement

Biomass is a fundamental parameter in thecharacterisation of microbial growth. Its measurement is

essential for kinetic studies on SSF. Direct determination

of biomass in SSF is very difficult due to the problem of se-

parating the microbial biomass from the substrate. This is

especially true for SSF processes involving fungi, because

the fungal hyphae penetrate into and bind tightly to the

substrate. On the other hand, for the calculation of growth

rates and yields, it is the absolute amount of biomass which

is important. Methods that have been used for biomass

estimation in SSF belong to one of the following catego-

ries.

Direct evaluat ion of b iomass

Complete recovery of fungal biomass is possible only under

artificial circumstances in membrane filter culture, because

the membrane filter prevents the penetration of the fungal

hyphae into the substrate (Mitchell et al, 1991). The whole

of the fungal mycelium can be recovered simply by peeling

it off the membrane and weighing it directly or after

drying. Obviously, this method cannot be used in actual

SSF. However, it could find application in the calibration

of indirect methods of biomass determination. Indirect

biomass estimation methods should be calibrated under

conditions as similar as possible to the actual situation in

SSF. The global mycelium composition could be estimated

through analysis of the mycelium cultivated in LSF in

conditions as close as possible to SSF cultivation.

Microscopic observations can also represent a good way to

estimate fungal growth in SSF. Naturally, optic examina-tion is not possible at high magnitude but only at stereo

microscope. Scanning Electron Microscope (SEM) is a

useful tool to observe the pattern of growth in SSF. New

approaches and researches are developed for image analy-

sis by computer software in order to evaluate the total

length or volume of mycelium on SEM photography. Ano-

ther new very promising approach is the Confocal Micro-

scopy, based on specific reaction of fungal biomass with

specific fluorochrome probes. Resulting 3D images of bio-

mass can open new ways to appreciate and measure bio-

mass in situ in a near future.

Since direct measurement of exact biomass in SSF is a very

difficult task, then other approaches have been preferred.

That for, the global stoichiometric equation of microbial

growth can be considered:preferred. That for, the global

stoichiometric equation of microbial growth can be

considered:

Carbon source + Water + Oxygen + Phosphorus + Nitrogen

Biomass + CO2 + Metabolites + Heat


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Raimbault, M.

181

Variation in each component is strictly related to the variation of others when all coefficients are maintained constants.

Then, measuring one of them allows to determine the evolution of the others.

Metabol ic measurement o f bio mass

- Respiratory metabolism Oxygen consumption and carbon dioxide release result

from respiration, the metabolic process by which aerobic

micro-organisms derive most of their energy for growth.

These metabolic activities are therefore growth associated

and can be used for the estimation of biomass synthesis.

As carbon compounds within the substrate are

metabolised, they are converted into biomass and carbon

dioxide. Production of carbon dioxide causes the weight of

fermenting substrate to decrease during growth, and the

amount of weight lost can be correlated to the amount of

growth that has occurred.

Growth estimation based on carbon dioxide release or

oxygen consumption assumes that the metabolism of thesecompounds is completely growth associated, which means

that the amount of biomass produced per unit of gas

metabolised must be constant. Sugama and Okazaki (1979)

reported that the ratio of mg CO2 evolved to mg dry

mycelia formed by Aspergillus oryzae on rice ranged from

0.91 to 1.26 mg CO2 per mg dry mycelium. A gradual

increase in this ratio was observed late in growth due to

endogenous respiration. Drastic changes can be observed

for the respiratory quotient which commonly changes with

the growth phase, i.e: germination, rapid and vegetative

growth, secondary metabolism, conidiation and degenera-

tion of the mycelium. Evolution of CO2 and O2 during SSFof Rhizopus on cassava is presented in fig 1.

Figure 1.- Kinetic evolution of CO2 and O2 in air flow during fermentation of Rhizopus on cassava.

The measurement of either carbon dioxide evolution or

oxygen consumption is most powerful when coupled with

the use of a correlation model. The term correlation model

is used here to denote a model that correlates biomass with

a measurable parameter. Correlation models are not

growth models as such, since they make no predictions as

to how the measurable parameter changes with time. The

usefulness of correlation models is that a biomass profile

can be constructed by following the profile of the

parameter during growth.

Application of these correlation models involving

prediction of growth from oxygen uptake rates or carbon

dioxide evolution rates requires the use of numerical tech-

niques to solve the differential equations. A computer and

appropriate software is therefore essential. If both the


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monitoring and computational equipment is available, then

these correlation models provide a powerful mean of

biomass estimation since continuous on-line measurements

can be made. Other advantages of monitoring effluent gas

concentrations with paramagnetic and infrared analysers

include the ability to monitor the respiratory quotient toensure optimal substrate oxidation, the ability to

incorporate automated feedback control over the aeration

rate, and the non-destructive nature of the measurement

procedure.

The metabolic activity in SSF is very important for stu-

dying all theoretical and practical aspects of respirometric

measurement of fungal biomass cultivated in SSF. Various

authors reported data concerning laboratory and scale up

experiments on respirometric measurement in several ap-

plications (Raimbault, 1981; Deschamps et al., 1982;

Bajracharya et al., 1980). An international training coursewas recently dedicated to advances and techniques to study

fungal growth on SSF (Raimbault et al., 1998).

- Production of extracellular enzymes or primary metaboli-

tes

Another metabolic activity that may be growth associated

is extracellular enzyme production. Okazaki and co-

workers (1980) claimed that the α-amylase activity wasdirectly proportional to mycelial weight for Aspergillus

oryzae grown in SSF on steamed rice. For growth of

Agaricus bisporus on mushroom compost, mycelial mass

was directly proportional to the extracellular laccase

activity for 70 days (Wood, 1979). In our works we havegenerally observed a good correlation between growth and

hydrolytic enzymes such as amylases, cellulases or pectina-

ses (Raimbault, 1981; Roussos et al., 1991b, 1993).

Also, we have frequently observed a good correlation bet-

ween mycelial growth and organic acid production, which

can be estimated from pH measurement or correlated by

HPLC analysis on extracts. In the case of Rhizopus, Soccol

(1992) demonstrated a close correlation between fungal

protein (biomass) and organic acids (citric, fumaric, lactic

or acetic).

Biomass Comp onents

The biomass can also be estimated from measurements of a

specific component, as long as the composition of the bio-

mass is constant and stable and the fraction of the

component representative.

Protein content:

The most readily measured biomass component is protein.

We used the protein content (as determined by the Lowry

method) to measure the growth rate of Aspergillus niger on

cassava meal (Raimbault and Alazard, 1980). Growth of

Chaetomium cellulolyticum on wheat straw was

determined from TCA insoluble nitrogen using the

Kjeldahl method (Laukevics et al., 1984); biomass protein

was then calculated as 6,25 times this value. In all cases

the protein content of the biomass was assumed to be

constant. Values of biomass protein content measured bythe Biuret method were consistent with those measured by

the Kjeldahl method. But, unfortunately, the Biuret method

was not suitable for application to SSF because of non-

specific interference by substrate starch. The Folin method

is more sensitive and allowed a greater dilution of the sam-

ple, which avoided interference from the starch in the

substrate. Therefore we have chosen the Folin technique to

measure protein enrichment in starchy substrates.

Nucleic acids

DNA content has been used to estimate the biomass of As- pergillus oryzae on rice (Bajracharya and Mudgett, 1980).

The method was calibrated using the DNA contents of

fungal mycelia obtained in submerged culture. DNA

contents were higher during early growth and then

decreased, levelling off as the stationary phase was

approached. The method was corrected for the DNA

content of rice, which remained unchanged since

Aspergillus oryzae did not produce extracellular DNases.

Methods based on DNA or RNA determination are reliable

only if there is little nucleic acid in the substrate and no in-

terfering chemicals are present.

Glucosamine

Glucosamine is a useful compound for the estimation of

fungal biomass, taking advantage of the presence of chitin,

poly-Nacetylglucosamine, in the cell walls of many fungi.

Interference with this method may occur when using com-

plex agricultural substrates containing glucosamine in

glucoproteins (Aidoo et al., 1981).

The accuracy of the glucosamine method for determination

of fungal biomass depends on establishing a reliable

conversion factor relating glucosamine to mycelial dry

weight (Sharma et al., 1977). However, the proportion of chitin in the mycelium will vary with age and the environ-

mental conditions. Mycelial glucosamine contents ranged

from 67 to 126 mg per g dry mycelium. Another disadvan-

tage of this method is the tedious extraction procedures

and processing times over 24 hours.

Ergosterol

Ergosterol is the predominant sterol in fungi. Glucosamine

estimation was therefore compared with the estimation of

ergosterol for the determination of growth of Agaricus

bisporus (Matcham et al, 1985). In solid cultures, directly

proportional relationships for glucosamine and ergosterol


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Raimbault, M.

183

against linear extension of the mycelium were obtained.

Determination of ergosterol was claimed to be more

convenient than glucosamine. It could be recovered and

separated by HPLC and quantified simply by

spectrophotometry, providing a sensitive index of biomass

at low levels of growth. HPLC was necessary to separatethe ergosterol from sterols endogenous to the solid

substrate. However, Nout et al., (1987) showed that the

ergosterol content of Rhizopus oligosporus varied from 2 to

24 micrograms per mg dry biomass, depending on the

culture conditions, aeration and substrate composition,

concluding that it was an unreliable method for following

growth.

Physical measurement of biomass

Peñaloza (1991) evaluated mycelial growth, based on the

difference in the electric conductivity between biomass andthe substrate. Good correlation with biomass was obtained

and a model was proposed.

Auria et al.,(1990) monitored the pressure drop in a pac-

ked bed during SSF of Aspergillus niger on a model solid

substrate consisting of ion exchange resin beads. Pressure

drop was closely correlated with protein production.

Pressure drop is a parameter that is simple to measure and

can be measured on-line. Further studies are required to

determine whether the use of pressure drop is generally

applicable for monitoring growth in SSF bioreactors under

forced aeration. An interesting point of this physical

technique resides in the fact that it is sensible to conidia-tion: early conidiophore stage makes the pressure to drop

drastically and a breaking point can be easily observed.

In conclusion, the measurement of biomass in SSF is

important to follow the kinetics of growth in relation to the

metabolic activity. Measurement of metabolic activity by

carbon dioxide evolution or oxygen consumption can be

generally applied, whereas extracellular enzyme produc-

tion will only be useful when enzyme production is

reasonably growth-associated.

Vital staining with fluorescein diacetate has potential in providing basic information as to the mode of growth of

fungi on complex solid surfaces, as this method can show

the distribution of metabolic activity within the mycelium.

But it cannot be measured on line.

On the other hand, in the production of protein enriched

feeds, the protein content itself is of greater importance

than the actual biomass concentration, and the variation in

biomass protein content during growth becomes less rele-

vant.

Overall, oxygen uptake and carbon dioxide evolution

methods are probably the most promising techniques for

biomass estimation in aerobic SSF as they provide on-line

information. The monitoring and computing equipment is

relatively expensive and will not be suitable for low

technology or rural applications. No method is ideally

suited to all situations, so the method most appropriate to a

particular SSF application must be chosen in each case on

the basis of simplicity, cost and accuracy. A good strategycan be a combination of several techniques based on the

determination of different parameters that can correlate

actual biomass with the material balance.

Environmental Factors

Environmental factors such as temperature, pH, water

activity, oxygen levels and concentrations of nutrients and

products significantly affect microbial growth and product

formation. In submerged stirred cultures, environmental

control is relatively simple because of the homogeneity of

the suspension of microbial cells and of the solution of nutrients and products in the liquid phase.

The low moisture content of SSF enables a smaller reactor

volume per substrate mass than LSF and also simplifies

product recovery (Moo-Young et a1., 1983). However,

serious problems arise with respect to mixing, heat exchan-

ge, oxygen transfer, moisture control and gradients of pH,

nutrient and product as a consequence of the heterogeneity

of the culture.

The latter characteristic of SSF render the measurement

and control of the above mentioned parameters difficult,

laborious and often inaccurate, thereby limiting the indus-trial potential of this technology (Kim et al, 1985). Due to

these problems, the micro-organisms that have been

selected for SSF are the more tolerant to a wide range of

cultivation conditions (Mudgett, 1986).

Moisture content and Water act iv i ty (Aw )

SSF process can be defined as microbial growth on solid

particles without the presence of free water. The water pre-

sent in SSF systems exists in a complexed form within the

solid matrix or as a thin layer either absorbed to the surfa-

ce of the particles or less tightly bound within the capillaryregions of the solid. Free water will only occur once the

saturation capacity of the solid matrix is exceeded.

However, the moisture level at which free moisture

becomes apparent varies considerably between substrates

and is dependant upon their water binding characteristics.

For example, free water is observed when the moisture

content exceeds 40% in maple bark and 50-55% in rice

and cassava (Oriol et al., 1988a). With most lignocellulosic

substrates free water becomes apparent before the 80%

moisture level is reached (Moo-Young et al., 1983).

The moisture levels in SSF processes, which vary between

30 and 85%, has a marked effect on growth kinetics, as


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184

shown on Figure 2 (Oriol et al., 1988b). The optimum

moisture level for the cultivation of Aspergillus niger on

rice was 40%, whereas on coffee pulp the level was 80%,

which illustrates the unreliability of moisture level as a

parameter for predicting microbial growth. It is now gene-

rally accepted that the water requirements of microorganisms should be defined in terms of the water

activity (Aw) rather than the water content of the solid sub-

strate. Aw is a thermodynamic parameter defined in

relation to the chemical potential of water. Aw is related to

the condensed phase of absorbed water, but it is well

correlated (less than 0.2 % error) to the relative humidity

(RH). Therefore: Aw = RH/100 = p/po, where p is the

vapour pressure of the water in the substrate and po is the

vapour pressure of pure water at the corresponding tempe-

rature, R being the ideal gas constant (Griffin, 1981). Aw

represents the availability of water for reaction in the solid

substrate.

The reduction of Aw has a marked effect on microbial

growth. Typically, a reduction in Aw extends the lag pha-

se, decreases the specific growth rate, and results in low

amount of biomass produced (Oriol et al., 1988b), asshown in fig.2. In general, bacteria require higher values

of Aw for growth than fungi, thereby enabling fungi to

compete more successfully at the Aw values encountered in

SSF processes. With the exception of halophilic bacteria,

few others grow at Aw values below 0.9 and most bacteria

require considerably higher minimum Aw values for

growth. Some fungi, on the other hand, stop growing only

at Aw values as low as 0.62 and a number of fungi used in

SSF processes have minimum growth Aw values between

0.8 and 0.9 .

The optimum moisture content for growth and substrate

utilisation is between 40 and 70% but depends upon the or-

ganism and the substrate used for cultivation. For example,

cultivation of Aspergillus niger on starchy substrates, such

as cassava (Raimbault and Alazard, 1980) and wheat bran

(Nishio et al., 1979), was optimal at moisture levels consi-

derably lower than on coffee pulp (Penaloza et al., 1991) or

sugarcane bagasse (Roussos et al., 1991a, 1991b). This is

probably because of the greater water holding capacity of

the latter substrate (Oriol et al., 1988b). The optimum Awfor growth of a limited number of fungi used in SSF pro-

cesses was at least 0.96, whereas the minimum Aw

required for growth was generally greater than 0 9. This

suggests that fungi used in SSF processes are not especially

xerophilic. The optimum Aw values for sporulation in

Trichoderma viride and Penicillium roqueforti were lower

than those for growth (Gervais et al., 1988). Maintenance

of the Aw at the growth optimum would allow fungal

biomass to be produced without sporulation.

Temperature and Heat Transfer


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Raimbault, M.

185

Stoichiometric global equation of respiration is highly exo-

thermic and heat generation by high levels of fungal activi-

ty within the solids lead to thermal gradients because of the

limited heat transfer capacity of solid substrates. In aero-

bic processes, heat generation may be approximated from

the rate or C02 evolution or O2 consumption. Each mole of C02 produced during the oxidation of carbohydrates relea-

ses 673 Kcal. Therefore, it is important to measure CO2evolution during SSF because it is directly related to the

risk of temperature increase. Detailed calculations of the

relation between respiration, metabolic heat and tempera-

ture were discussed in early works on SSF with Aspergillus

niger growing on cassava or potato starch (Raimbault,

1981). The overall rate or heat transfer may be limited by

the rates of intra- and inter-particle heat transfer and by

the rate at which heat is transferred from the particle surfa-

ce to the gas phase. The thermal characteristics of organic

material and the low moisture content in SSF areespecially difficult conditions for heat transfer. Saucedo-

Castañeda et al., l990 developed a mathematical model for

evaluating the fundamental heat transfer mechanism in

static SSF and more specifically to assess the importance of

convection and conduction in heat dissipation. This model

can be used as a basis for automatic control of static

bioreactors.

Heat removal is probably the most crucial factor in large

scale SSF processes. Conventional convection or

conductive cooling devices are inadequate for dissipating

metabolic heat due to the poor thermal conductivity of

most solid substrates and results in unacceptable

temperature gradients. Only evaporative cooling devices provide sufficient heat elimination capacity. Although the

primary function of aeration during aerobic solid state

cultivations was to supply oxygen for cell growth and to

flush out the produced carbon dioxide, it also serves a

critical function in heat and moisture transfer between the

solids and the gas phase. The most efficient process for

temperature control is water evaporation.

Maintaining constant temperature and moisture content

simultaneously in large scale SSF is generally difficult, but

using the proper ancillary equipment can do this. The

reactor type can have a large influence on the quality of

temperature control achieved. It depends highly of the typeof SSF: static on clay or vertical exchangers, drums or me-

chanically agitated.

Control of pH and r isks of con taminat ion

The pH of a culture may change in response to metabolic

activities. The most obvious reason is the secretion of

organic acids such as citric, acetic or lactic, which will

cause the pH to decrease, in the same way than ammonium

salts consumption. On the other hand, the assimilation of

organic acids which may be present in certain media will

lead to an increase in pH, and urea hydrolysis will result in

alkalinisation. The kinetics of pH variation depends high-

ly on the microorganism. With Aspergillus sp., Penicillium

sp. , and Rhizopus sp. the pH can drop very quickly below

3.0; for other types of fungi, like Trichoderma, Sporotri-

chum, Pleurotus sp. the pH is more stable between 4 and 5.

Besides, the nature of the substrate has a strong influence

on pH kinetics, due to the buffering effect of lignocellulosicmaterials.

We have used a mixture of ammonium salt and urea to

control the pH decrease during growth of A. niger on star-

chy substrates (Raimbault, 1981). A degree of pH control

may be obtained by using different ratios of ammonium

salts and urea in the substrate. Hydrolysis of urea liberates

ammonia, which counteracts the rapid acidification

resulting from uptake of the ammonium ion (Raimbault

and Alazard. 1980). In this manner, we obtained optimal

growth of Aspergillus niger on granulated cassava meal

when using a 3:2 ratio (on a nitrogen basis) of ammoniumto urea. We observed that during the first stage of cultiva-

tion the pH increased as the urea was hydrolysed. During

the subsequent rapid growth stage, ammonium

assimilation exceeded the rate of urea hydrolysis and the

pH decreased, but increased again in the stationary phase.

During cultivation, the pH remained within the limits of

pH 5 to pH 6.2, whereas a lower urea concentration

resulted in a rapid decrease in pH.

In a similar way, pH adjustment during pilot plant

cultivation of Trichoderma viride on sugar-beet pulp was

effective by spraying with urea solutions due to the urease

activity of the micro-organism that caused an increase in

pH by producing ammonia (Durand et al., 1988).

Finally, in fungal or yeast SSF, bacterial contamination

may be minimised or prevented by employing a suitably

low pH.

Oxygen uptake

Aeration fulfils four main functions in SSF, namely (i) to

maintain aerobic conditions, (ii) to desorb carbon dioxide,

(iii) to regulate the substrate temperature and (iv) to

regulate the moisture level. The gas environment may

significantly affect the relative levels of biomass andenzyme production. In aerobic LSF oxygen supply is often

the growth limiting factor due to the low solubility of

oxygen in water. In contrast, a solid state process allows

free access of atmospheric oxygen to the substrate.

Therefore, aeration may be easier than in submerged

cultivations because of the rapid rate of oxygen diffusion

into the water film surrounding the insoluble substrate

particles, and also because of the very high surface of

contact between gas phase, substrate and aerial mycelium.

The control of the gas phase and air flow is a simple and

practical mean to regulate gas transfer and generally no

oxygen limitation is observed in SSF when the solid sub-

strate is particular. It is important to maintain a good ba-


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186

lance between the three phases in SSF (Auria, 1990; Sau-

cedo-Castañeda et al., 1990).. By this very simple aeration

process, it is also possible to induce metabolic reactions, ei-

ther by water stress, heat stress or temperature changes, all

processes that can drastically change biochemical or

metabolic behaviour.

Concluding Remarks

SSF is a well-adapted process for cultivation of fungi on

vegetal materials which are breakdown by excreted hydro-

lytic enzymes. In contrast with LSF where water is in large

excess, water activity is a limiting factor in SSF. On the

other hand, oxygen is a limiting factor in LSF but not in

SSF, where aeration is promoted by the porous and parti-

cular structure and by the high surface are of contact which

facilitate mass transfer between gas and liquid phases.

SSF are aerobic processes where respiration is fundamental

for energy supply but, because respiratory metabolism is

highly exothermic, severe limitation of growth can occur

when heat transfer is not efficient enough to avoid tempe-

rature increase.

This is why it is so important to study and control respiro-

metry in SSF. We have developed a laboratory technique to

measure CO2 and O2 on line in SSF.

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